Our long-term aim is to elucidate the cellular and molecular events underlying the initiation of hematopoiesis in the mammalian embryo. While definitive hematopoiesis in the fetus and adult is multilineage, hematopoiesis in the yolk sac has been widely considered to consist exclusively of the primitive erythroid lineage that generates nucleated red cells. During the initial funding period, we defined two temporally distinct primitive and definitive waves of hematopoiesis that originate in the yolk sac of the mouse embryo. Furthermore, our studies indicate that primitive hematopoiesis in mammals is multilineage, consisting not only of primitive erythroid and macrophage progenitors but also of embryonic megakaryocyte progenitors that have different growth requirements when compared to their bone marrow counterparts. In the first aim of this renewal, we will investigate the lineage relationships that exist among the hematopoietic progenitors that arise during early gastrulation, focusing particular attention on the embryologic origin and ontogeny of the megakaryocyte lineage. Primitive red cells originate in close association with endothelial cells in yolk sac blood islands, suggesting that they arise from a common "hemangioblast" precursor. Recently, in collaboration with G. Keller (Mt. Sinai School of Medicine, NY), I identified a unique precursor with hematopoietic and endothelial potential in the early mouse embryo. In the second aim of this renewal, I will continue to investigate the potential and ontogeny of embryonic hemangioblasts. Notch signaling has been increasingly implicated in the differentiation of the hematopoietic system in the adult and is necessary for vascular development in the yolk sac. We hypothesize that Notch signaling regulates the establishment of the hematopoietic and endothelial systems in the mammalian embryo. In Aim 3 of this proposal, we will use both Notch activation and inhibition approaches in primary yolk sac explants and ES cell-derived embryoid bodies to begin to delineate the role of the Notch pathway in embryonic hematopoiesis. A better understanding of the initiation of mammalian hematopolesis, and its regulation by Notch signaling, will ultimately provide insights into the ontogeny, regulation and expansion of hematopoietic stem cells, as well as the origin of leukemias and bone marrow failure syndromes. This will ultimately lead to improvements in bone marrow transplantation for the curative treatment of congenital anemias, genetic diseases and several forms of childhood and adult cancers.